Biomedical subjects
Publications and source records attributed to T Barnard.
The healthcare environment is changing rapidly. Work reorganization and redesign can lead to department and unit closures, resulting in work transitions for many staff members. The authors describe the findings of focus groups held with staff and management on units that have closed. These interviews highlight the difficulties encountered with change and provide suggestions to reduce uncertainty during department or unit closures.
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It has been realized almost from the start of biological electron microscopy that the use of low temperature in specimen preparation might alleviate the problems associated with conventional chemical fixation and plastic embedding. In practice it has been very difficult to realize any benefits, owing to the problems encountered in trying to prepare samples with the water transformed to ice, yet without the sample itself hopelessly distorted by ice crystals. The difficulty is caused by the poor diffusibility of heat through water, the main constituent of almost all biological samples. Even with a maximal temperature gradient between the sample surface and the coolant, and a perfect contact between the two, heat diffusibility through the sample quickly becomes the limiting parameter affecting cooling rate. Unless the cooling rate is as high as 10(2) or even 10(3) degrees C.msec.-1, the ice formed will be crystalline. Such high cooling rates can only be attained in the first few micrometers of even very small samples: therefore, for bulk samples one must either accept the use of a 'suitable' cryoprotectant, or else accept the presence of ice crystals. Theoretical and practical considerations for freezing samples using liquid and solid coolants will be reviewed. Special consideration will be given to the present status of cryoprotection and the indications and contra-indications for its use.
The effect of nortriptyline against ventricular arrhythmias was determined in 16 cardiac patients with 30 or more ventricular premature depolarizations per hour. Nortriptyline was administered orally, 0.5 mg/kg body weight per day, and increased by 0.5 mg/kg per day every third day until ventricular premature depolarizations were suppressed (greater than or equal to 80%), adverse effects occurred or a total daily dose of 3.5 mg/kg per day was given. Each patient had daily 24 hour continuous electrocardiograms, 12 lead standard electrocardiograms and physical examination; blood pressure was measured in the supine and standing position four times a day. Each patient also had radionuclide angiography at rest to measure ejection fraction before and at the effective or maximal dose. Thirteen patients (81%) had an antiarrhythmic response and 11 met the study criterion of at least 80% improvement. Doses ranged from 50 to 200 mg/day (mean 111 +/- 45), steady state plasma concentration ranged from 46 to 410 ng/ml (mean 153 +/- 96) and half-life of elimination of nortriptyline was 4 to 22 hours (mean 13 +/- 4). Administration of nortriptyline did not depress mean ejection fraction (before 42 +/- 12%, after 41 +/- 12%); it was associated with an orthostatic decrease in systolic blood pressure (mean -13 +/- 13 mm Hg). Nortriptyline is an effective antiarrhythmic agent which may be given twice a day even in patients with impaired ventricular function.
Pancreatic acinar cells are thought to secrete a fluid containing digestive enzymes and electrolytes and use e.g. calcium as a second messenger upon stimulation. Together with their pronounced morphological polarity, they provide a model system to study the effect of different preparation methods for quantitative biological electron probe X-ray microanalysis (EPXMA) of ultrathin sections. Several preparation methods i.e., freeze-drying and plastic-embedding, freeze-substitution (2 days) and freeze-drying of ultrathin cryosections have been applied to examine the retention of sodium, magnesium, phosphorus, sulfur, (chlorine), potassium and calcium in subcellular compartments (basal cytoplasm, apical cytoplasm, mitochondria and zymogen granules). In freeze-substituted samples the phosphorus, potassium and sulfur concentrations were 2-3 times lower in all compartments compared to freeze-dried, plastic-embedded samples. Intracellular potassium-to-sodium ratios obtained on frozen substituted and frozen-dried, plastic-embedded samples were considerably lower than for cryosections. Element gradients between adjacent organelles were large in frozen-dried cryosections, smaller in frozen-dried plastic- embedded samples and insignificant in frozen-substituted samples.
Biological X-ray microanalysis of diffusible elements within cellular and subcellular compartments requires preparation methods to retain electrolytes in the compartments they occupied in vivo. X-ray microanalysis of frozen-dried, plastic-embedded samples has been used to quantitate electrolytes at the cellular level. We have compared the subcellular elemental distribution in dry cut sections from such samples with that in ultrathin frozen-dried cryosections. Rat pancreases were quench-frozen onto a helium-vapor-cooled copper block. Cryosections were cut at 130-150 K, transferred using a Gatan cold stage, frozen-dried in the column and analysed at 190 K. Tissue fragments were frozen-dried at 190 K, and cut on a dry knife at 293 K. Both samples provided images permitting unambiguous identification of all major compartments except the Golgi complex. Intracellular potassium-to-sodium ratios obtained on frozen-dried plastic-embedded sections were lower than for cryosections (e.g. 1.77 in basal cytoplasm in plastic sections as compared to 4.34 for cryosections) and varied with the pre-embedding procedure (e.g. 1.77 in formaldehyde-fixed as compared to 2.87 in osmium-fixed plastic sections). Potassium gradients between adjacent organelles were large in cryosections and insignificant in plastic-embedded material. Higher cytoplasmic phosphorus, potassium and sulfur concentrations were observed in cryosections. Therefore, a redistribution of electrolytes and covalently bound elements occurred subcellularly in the plastic sections. Calcium was quantifiable in most organelles in cryosections but the plastic lowered sensitivity too much to permit routine calcium quantification. We conclude that in our hands frozen-dried, plastic-embedded samples were compromised and provided lower sensitivity than cryosections.(ABSTRACT TRUNCATED AT 250 WORDS)
Cultured rat hepatocytes were incubated in medium containing 1.0 mM oleic acid. The incorporation of [3H]glycerol into cell-associated and medium triacylglycerols was measured after 2 h incubation. More than 95% of the secreted [3H]triacylglycerols were recovered in the very low density lipoprotein (VLDL) fraction (d less than 1.006). Chloroquine and other lysosomotropic amines promoted a marked decrease in [3H]triacylglycerol secretion from the hepatocytes while the synthesis was unaffected. At 50-200 microM final concentration, chloroquine inhibited secretion of triacylglycerols by 70-90% of the control. Similar results were obtained when the mass of secreted triacylglycerols was measured. Chloroquine caused decreased secretion of [3H]triacylglycerols after 15-30 min incubation and the inhibitory effect was completely reversible within 1-2 h after washout of chloroquine. The reduced triacylglycerol secretion was not due to increased reuptake of secreted lipoproteins or decreased protein synthesis caused by chloroquine. Electron microscopy of chloroquine-treated cells showed that the inhibition of VLDL secretion occurs at or prior to the level of the Golgi apparatus. These results suggest that chloroquine interferes with crucial steps in the secretory process and/or that lysosomal function could be essential for secretion of VLDL.
The preparation of a set of aminoplastic standards for quantitation of sodium, magnesium, phosphorus, sulfur, chlorine, potassium, and calcium in ultrathin plastic sections of soft tissue is described. The standards are low in cost and easy to prepare and handle. They cover concentrations up to 750 mmoles/kg dry weight, and display chemical and physical properties similar to those plastic-embedded samples. The standards can be used to convert peak-to-continuum ratios obtained from the specimen to elemental concentrations. An application of these standards is shown using rat exocrine pancreas as a model. The standards can also be used to advantage for the calibration of the detector efficiency.
The application of X-ray microanalysis to problems of cell physiology required the development of methods to retain diffusible substances within the subcellular compartments that they occupied in vivo. Several groups have developed methods of rapidly freezing small samples in ways that minimize mechanical traumae and ice crystal formation. This provides a narrow zone from which cryosections, believed to be representative of the in vivo distribution of electrolytes, can be cut. The production of thin (less than 0.5 micrometers) cryosections that are apparently free of diffusion can be routinely performed when temperature parameters are kept below 173 K. Efficient cryosectioning requires several modifications to commercially available machines, in order to improve the ease and reliability with which various manipulations can be carried out. Initial attempts to localize calcium at the subcellular level were disturbed by the use of mechanically damaged specimens and by insufficiently cold conditions in the cryochamber. Such sections indicated that mitochondria were calcium-rich organelles. When tissue freezing and cryosectioning were performed under optimized conditions, mitochondrial calcium was so low as to be quantifiable only with difficulty. Available microanalytical results show that ER-rich cytoplasm and terminal cisternae of the sarcoplasmic reticulum seem to contain higher levels of calcium than mitochondria. Nuclei and secretory granules also contain more calcium than mitochondria.
Calcium and magnesium distribution in mammalian exocrine glands under resting, stimulated and pathological conditions was investigated by X-ray microanalysis of thick and ultrathin cryosections. Ultrathin sections were cut from tissue frozen in the presence of a polymer cryoprotectant, dextran. The effect of this treatment on isolated rabbit pancreas. Dextran caused a disturbance in water and ion transport, partly due to an osmotic effect and the impermeability of the pancreatic epithelium to dextran; this does, however, not necessarily invalidate intracellular measurements on frozen-dried sections. Cholinergic stimulation of the rat pancreas caused a change of Ca distribution from the basal to the apical part of the cell; this may be a component of the secretory Ca flux. Kinetic considerations make a significant Ca movement via the ER-Golgi endomembrane space less likely. The mitochondrial Ca concentration is low, and not significantly changed by cholinergic stimulation. X-ray microanalysis was carried out on submandibular glands of rats after chronic treatment with reserpin and/or isoproterenol (an animal model for cystic fibrosis, CF). The acinar cells had elevated Mg and Ca and lowered K concentrations. Analysis of ultrathin cryosections showed high levels of Ca and Mg in secretory granules, mucus globules and the ER. Ca and Mg in the ER may be transported intracellularly with secretory proteins to secretion granules or mucus globules. The decrease in cell K may be due to efflux of K caused by elevated cytoplasmic Ca levels. A similar decrease in cell K was caused by incubation of rat salivary glands with diluted serum from CF patients, a treatment which has been reported to mimic the effect of a rise in cytoplasmic Ca.
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The ultrastructural changes caused by incubation of rat liver and brown fat in buffered solutions of high molecular weight cryoprotectives (Dextran and polyvinyl pyrrolidone) at high concentrations (up to 25% w/v) have been examined. Under appropriate conditions of incubation rather small qualitative changes were found. Hepatocytes showed some signs of plasmolysis. Evidence for the endocytosis of Dextran and polyvinyl pyrrolidone was obtained. Cryoultramicrotomy of unfixed, quench-frozen specimens after the same incubation procedures showed good cutting properties and only slight ice-crystal damage. Several of the larger tissue compartments were recognizable in such sections. Although high molecular weight cryoprotectants do penetrate cells by endocytosis (and these effects require further functional evaluation), the present observations provide further support for the idea that such compounds significantly improve the quench-freezing of biological specimens and offer a practical way forward for the preparation of material for X-ray microanalysis of diffusible elements.
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Clusters of dense particles were observed irregularly in mitochondrial profiles of shock-frozen brown adipose tissue and liver when cryosectioned at a chamber temperature of -80 degrees C and not if sectioning was performed at -110 degrees C. The particles are rich in Ca and P (Ca/P =0.5); they are therefore not any form of tricalcium orthophosphate (e.g. hydroxyapatite). They apparently form by electrolyte diffusion in the section and therefore cannot be considered as representing the in vivo pool of mitochondrial calcium.
The appearance of ultrathin, dry-cut cryosections of brown adipose tissue and liver was found to be strongly dependent upon adequate freeze-drying. If freeze-drying was inadequate, diffusion of substances could be demonstrated and freezing damage was not apparent. Diffusion was manifested as an electron dense film over structural features such as triglyceride droplet profiles and the edges of the section; when this film was thick, X-ray signals for P, S, Cl, K and Ca could be detected from it, in different proportions to those found in the section. The frequency and intensity of diffusion were lowered by decreasing the temperature in the cryochamber from about 200 K +/- 5 K to 163 K +/- 5 K by forced evaporation of liquid N2 using an extra heater. The lowest incidence of diffusion was obtained in conjunction with this device, by leaving the sections in the cryochamber for at least 2 h during drying, either over a drying agent or under moderate vacuum. Such sections showed a narrow zone (A) at the edge that lacked obvious ice-crystal damage, a thicker zone (B) of moderate ice-crystal damage and the bulk of the interior (zone C) severely damaged by freezing. Qualitatively different, reproducible X-ray spectra could be obtained from ultrastructural features even in areas showing some signs of diffusion.
Dark-gold sections of osmium tetroxide-fixed, Epon-embedded brown adipose tissue before and after low-temperature oxygen plasma microincineration were examined using a high-resolution scanning transmission electron microscope and an energy-dispersive X-ray spectrometer. Microincineration produced ash patterns which were free of organic matrix, chlorine (from the Epon) and probably osmium (from the fixative). X-ray sensitivity was improved by a factor of 2-4 owing to decreased background, and sulphur, calcium and probably phosphorus were detected in the ash. Fidelity of the ash patterns permitted microanalytical spatial resolution of 0.1 micrometer or better. Oxygen plasma microincineration is thus shown to offer advantages for high resolution X-ray microanalysis of conventionally sectioned biological material. Its future application to shock-frozen, frozen-dried, unstained sections is indicated.